Novel PE-DT-fusion toxin enhances internalization and target-dependently restores the cytotoxicity of recombinant...

Recombinant immunotoxins (RITs) are fusion proteins of a targeting domain, such as an antibody fragment, and a truncated toxin, including Pseudomonas exotoxin A (PE) or diphtheria toxin (DT). Limiting their possible fusion partners, the targeting domain of DT is typically fused C-terminally and that of PE is fused N-terminally. Among other factors, the activity of immunotoxins depends on the target antigen and the target-specific intracellular trafficking. Because a novel anti-CD138-PE immunotoxin was inactive against multiple myeloma, we hypothesized that rational toxin design would improve trafficking and, thus, cytotoxicity.

We, therefore, generated several variants with distinct domain sequences including the catalytic (C) units of PE and DT, the furin-cleavage site of PE (fu), the transport (T) domain of DT, and the ER retention sequence KDEL. The lead candidate-a fusion of PE and DT-was combined with antibodies against CD138, CD22, glypican-3, or mesothelin.

This final toxin moiety was internalized 4-fold more efficiently over time on average compared to PE-based RITs, regardless of the target antigen or cell type. Improved internalization of DT over PE did not depend on a specific domain of DT. Instead, it was competed dose-dependently by poly-D lysine (PDL), indicating a more unspecific charge effect of DT over PE. The improved internalization frequently translated to enhanced cytotoxicity. Finally, combinatorial treatment with actinomycin D demonstrated synergistic effects with the lead PE-DT-fusion toxin.

This novel and target-dependently more potent toxin moiety may provide a framework for future immunotoxin design, possibly widening the range of target antigens, including the ongoing efforts to enhance mesothelin- or CD138-targeting immunotoxins against cancer or autoimmune diseases.